Detection of malaria

ABSTRACT

A method of detecting falciparum, tertian, quartan and ovale malaria parasites by using primers represented by the sequences (SEQ ID NO:1), (SEQ ID NO:3) to (SEQ ID NO:10): 
     
         5&#39;AAGTCATCTTTCGAGGTGAC3&#39;                                   (SEQ ID NO:4) 
    
     
         5&#39;GAATTTTCTCTTCGGAGTTTA3&#39;                                  (SEQ ID NO:5) 
    
     
         5&#39;GAGACATTCTTATATATG3&#39;                                     (SEQ ID NO:3) 
    
     
         5&#39;GAAAATTCCTTTCGGGGA3&#39;                                     (SEQ ID NO:1) 
    
     
         5&#39;CGACTAGGTGTTGGATGA3&#39;                                     (SEQ ID NO:6) 
    
     
         5&#39;GAACGAAAGTTAAGGGAGT3&#39;                                    (SEQ ID NO:7) 
    
     
         5&#39;ACTGAAGGAAGCAATCTAA3&#39;                                    (SEQ ID NO:8) 
    
     
         5&#39;TCAGATACCGTCGTAATCTT3&#39;                                   (SEQ ID NO:9) 
    
     
         5&#39;CCAAAGACTTTGATTTCTCAT3&#39;                                  (SEQ ID NO:10) 
    
     This invention allows all of the plasmodia, which infect the human, to be detected easily, rapidly and with a high sensitivity or distinguished accurately from one another, thus permitting treatment for malaria and large-scale mass examination in the area where malaria is prevalent.

TECHNICAL FIELD

The present invention relates to a nucleotide fragment specific for anovale malaria parasite (Plasmodium ovale) and/or a quartan malariaparasite (Plasmodium malariae) and a method of detecting a falciparummalaria parasite (Plasmodium falciparum), a tertian malaria parasite(Plasmodium vivax), a quartan malaria parasite and/or an ovale malariaparasite at the same time or in distinction from one another making gooduse of this nucleotide fragment.

BACKGROUND ART

Malaria is an infectious disease caused by the infection of a plasmodiumand is carried by Anopheles mosquitoes. Malaria not only widely spreadsall over the tropics, but also occurs in many of temperate regions.According to the report of World Health Organization (WHO), the putativenumbers of cases and deaths amount to two hundred and seventy millionand two million a year, respectively World Health Organization: Malaria,"Tropical Disease in Research, 1989-1990" (8) UNDP/World Bank/WHO (TDR),WHO Geneva (1991), p. 29-40!. It is hence of urgent necessity toconsider countermeasures. The countermeasures are centered on earlydetection and early treatment by mass examination. However, the presentdiagnosis does not cope with wide-range mass examination.

The microscopy in which a blood smear is Giemsa-stained and the stainedsample is then observed through a microscope has heretofore been used inthe diagnosis of malaria. The microscopy is cheap and can be simplypracticed. On the other hand, its judgment requires much skill. Sincethe number of samples on which one inspection expert can test in a dayis 60 to 70 specimens, such a method cannot cope with a great number ofinfected persons in the area where malaria is prevalent and the numberof inspection experts is insufficient. In particular, when targets aresubclinical persons as in mass examination, many of positive personshave a very few plasmodia in their blood. Therefore, it takes a longtime to judge, and there is a great risk of making a wrong diagnosis asto the presence of a plasmodium and the kind of the plasmodium ifpresent.

The serodiagnosis is widely practiced after the microscopy. Theserodiagnosis is a method in which an antibody (which is said to lightenthe symptoms upon infection though its activity is not so high that theinfection is prevented) produced after infected with a plasmodium isdetected, and techniques such as the indirect fluorescent antibodytechnique and ELISA (enzyme linked immunosorbent assay) have beendeveloped. These methods permit the treatment of a large number ofsamples and are hence being widely used in mass examination and as meansfor immunological research studies even at present. However, such amethod involves a problem in that even if the antibody is found to bepositive by the serodiagnosis, this detection cannot distinguishinfection at the time of the inspection from infection before thenBruce-chwatt, L. J., Lancet 2,1509-1511 (1987)!. This method isclinically considered to be an ancillary diagnosis.

With the recent development of the molecular biology, it has also beenconducted to detect plasmodia by DNA diagnosis. It has been reportedthat characteristic sequences consisting of many repetitions having alength of 20 to 30 bases exist in genes of plasmodia, probes specificfor these regions as targets have been developed, which can achievestill higher sensitivity than that of probes making any other region asa target Robert, H., Science 231, 1434-1436 (1986)!. However, thismethod has involved problems such as the detection requireshybridization, which makes use of a membrane and is hence complicated inoperation, and the sensitivity is lower than expected when the method isclinically tested in practice Lamar, D. E., Am. J. Trop. Med. Hyg. 34,663-667 (1985)!. In order to enhance the sensitivity, a detection methodmaking use of a gene-amplifying technique has thus been developed Molec.Cell. Probes 4, 409-414 (1990)!. This method is a method in which a partof a 18S ribosome RNA gene of a falciparum malaria parasite is subjectedto gene amplification to detect it.

However, since an area where falciparum malaria is prevalent oftenoverlap an area where tertian malaria is prevalent, the detection of thefalciparum malaria parasite alone according to the above-describedmethod is insufficient to conduct effective treatment. Even ifhigh-sensitive detection can be realized by the gene-amplifyingreaction, the operation for the detection remains complicated.Accordingly, this method is far from the practical use as a means formass examination under the circumstances.

In view of the foregoing circumstances, the present inventors found anucleotide fragment specific for a falciparum malaria parasite, anucleotide fragment specific for a tertian malaria parasite and anucleotide fragment specific for both malaria parasites, and a method ofdetecting a falciparum malaria parasite and/or a tertian malariaparasite making good use of these nucleotide fragments, and previouslyfiled an application for patent (Japanese Patent Application No.36485/1992).

However, as plasmodia which infect the human, an ovale malaria parasiteand a quartan malaria parasite are known in addition to theabove-described plasmodia of two species. With respect to these fourkinds of malaria, considerable skill is required to distinguish themfrom one another by morphological observation through a microscopy. Inparticular, the tertian malaria parasite and the ovale malaria parasiteare extremely similar to each other in both morphology and clinicalsymptoms when infected with them and hence are very difficult to bedistinguished from each other.

It is accordingly an object of the present invention to provide anucleotide fragment specific for an ovale or quartan malaria parasite,by which only the ovale or quartan malaria parasite can be detected, anda method of detecting four species of plasmodia at the same time or indistinction from one another making good use of this nucleotidefragment.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided a nucleotidefragment comprising a base sequence (SEQ ID NO:1) represented by thefollowing sequence (SEQ ID NO:1), a base sequence complementary theretoor a mutation sequence thereof; and a primer or probe for detecting anovale malaria parasite comprising this nucleotide fragment.

    5'GAAAATTCCTTTCGGGGA3'                                     (SEQ ID NO:1)

According to the present invention, there is also provided a nucleotidefragment comprising a base sequence (SEQ ID NO:2) represented by thefollowing sequence (SEQ ID NO:2), a base sequence complementary theretoor a mutation sequence thereof; and a primer or probe for detecting anovale malaria parasite comprising this nucleotide fragment. ##STR1##

According to the present invention, there is further provided anucleotide fragment comprising a base sequence (SEQ ID NO:3) representedby the following sequence (SEQ ID NO:3), a base sequence complementarythereto or a mutation sequence thereof; and a primer or probe fordetecting a quartan malaria parasite comprising this nucleotidefragment.

    5'GAGACATTCTTATATATG3'                                     (SEQ ID NO:3)

According to the present invention, there is still further provided amethod of distinguishing falciparum malaria, tertian malaria, quartanmalaria and ovale malaria from one another comprising using, as probes,nucleotide fragments separately comprising base sequences (SEQ ID NOS:1, 3, 4, 5, and 6, respectively) respented by the following sequences(SEQ ID NOS:1, 3, 4, 5 and 6, base sequences complementary thereto ormutation sequences thereof.

    5'AAGTCATCTTTCGAGGTGAC3'                                   (SEQ ID NO:4)

    5'GAATTTTCTCTTCGGAGTTTA3'                                  (SEQ ID NO:5)

    5'GAGACATTCTTATATATG3'                                     (SEQ ID NO:3)

    5'GAAAATTCCTTTCGGGGA3'                                     (SEQ ID NO:1)

    5'CGACTAGGTGTTGGATGA3'                                     (SEQ ID NO:6)

According to the present invention, there is yet still further providedprimers for detecting plasmodia comprising a nucleotide fragmentcomprising any one of base sequences (SEQ ID NOS:7-10, respectively)represented by the following sequences (SEQ ID NOS:7-10) or mutationsequences thereof.

    5'GAACGAAAGTTAAGGGAGT3'                                    (SEQ ID NO:7)

    5'ACTGAAGGAAGCAATCTAA3'                                    (SEQ ID NO:8)

    5'TCAGATACCGTCGTAATCTT3'                                   (SEQ ID NO:9)

    5'CCAAAGACTTTGATTTCTCAT3'                                  (SEQ ID NO:10)

When the nucleotide fragments according to the present invention, whichhave the above-mentioned base sequences, respectively, are used as aprimer or primers either singly or in various combinations with oneanother, all of the plasmodia, which infect the human, namely, afalciparum malaria parasite, a tertian malaria parasite, a quartanmalaria parasite and an ovale malaria parasite, can be detected easily,rapidly and with a high sensitivity at the same time or distinguishedaccurately from one another. Therefore, they are extremelyadvantageously used in treatment for malaria and large-scale massexamination in the area where malaria is prevalent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the comparison of partial sequences of 18S ribosomeRNA genes of a falciparum malaria parasite, a tertian malaria parasite,a quartan malaria parasite, an ovale malaria parasite and the human (SEQID NO:14-18). In the drawing, - means deletion. Numerals in parenthesesindicate base sequence numbers. Underlines indicate regions respectivelycorresponding to primers or probes (SEQ ID NOS:1, 3, 4 and 5 ! specificfor the respective plasmodia. 1, 4, 2 and 5 indicate regionsrespectively corresponding to primers represented by (SEQ ID NOS:1and3-10). 3 indicates a region corresponding to a common probe representedby the sequence (SEQ ID NO:6).

BEST MODE FOR CARRYING OUT THE INVENTION

The nucleotide fragments represented by the sequences (SEQ ID NOS:1 and3-10) in the present invention can be classified in the following manneraccording to their characteristics.

(A) A nucleotide fragment selectively detecting or amplifying only the18S ribosome RNA gene of the ovale malaria parasite.

    5'GAAAATTCCTTTCGGGGA3'                                     (SEQ ID NO:1)

(B) A nucleotide fragment selectively detecting or amplifying only the18S ribosome RNA gene of the quartan malaria parasite.

    5'GAGACATTCTTATATATG3'                                     (SEQ ID NO:3)

(C) A nucleotide fragment selectively detecting or amplifying only the18S ribosome RNA gene of the falciparum malaria parasite.

    5'AAGTCATCTTTCGAGGTGAC3'                                   (SEQ ID NO:4)

(D) A nucleotide fragment selectively detecting or amplifying only the18S ribosome RNA gene of the tertian malaria parasite.

    5'GAATTTTCTCTTCGGAGTTTA3'                                  (SEQ ID NO:5)

(E) Nucleotide fragments detecting or amplifying all of the 18S ribosomeRNA genes of the falciparum malaria parasite, tertian malaria parasite,quartan malaria parasite and ovale malaria parasite.

    5'CGACTAGGTGTTGGATGA3'                                     (SEQ ID NO:6)

    5'GAACGAAAGTTAAGGGAGT3'                                    (SEQ ID NO:7)

    5'ACTGAAGGAAGCAATCTAA3'                                    (SEQ ID NO:8)

    5'TCAGATACCGTCGTAATCTT3'                                   (SEQ ID NO:9)

    5'CCAAAGACTTTGATTTCTCAT3'                                  (SEQ ID NO:10)

All of these nucleotide fragments can be chemically synthesized by meansof a DNA synthesizer. The base sequence of the nucleotide fragment of(SEQ ID NO:1) was determined in the following manner. That is, blood wasfirst collected from a patient infected with an ovale malaria parasite.DNA obtained from this was subjected to a gene-amplifying reaction usingthe primers of (SEQ ID NO:7-8). As a result, an amplified product of 140bp was obtained. The base sequence of the amplified product wasdetermined and found to be the following sequence (SEQ ID NO:2):##STR2##

Of these nucleotide fragments, the nucleotide fragment having (SEQ IDNO:1) does not exist in the plasmodia of falciparum malaria, tertianmalaria and quartan malaria and selectively detects or amplifies the 18Sribosome RNA gene of the ovale malaria parasite.

It was also confirmed that the nucleotide fragment of (SEQ ID NO:3) issynthesized by the DNA synthesizer and selectively detects or amplifiesthe 18S ribosome RNA gene of the quartan malaria parasite by agene-amplifying reaction. In a similar manner, the reactivity of thenucleotide fragments of (SEQ ID NO:4-6) was also confirmed.

With respect to the mutation sequences in the nucleotide fragmentsaccording to the present invention, any mutation sequence may bementioned so far as it functions as a primer or probe for detecting afalciparum malaria parasite, a tertian malaria parasite, a quartanmalaria parasite and/or an ovale malaria parasite. Examples thereofinclude those in which part of bases are deleted from the original basesequences represented by (SEQ ID NOS:1-10) to, and those obtained byreplacement or by addition of other bases. More specifically, there maybe mentioned base sequences corresponding to their corresponding regionsof genes of the mutated strains of the above-described plasmodia, RNAsequences corresponding to these genes and the like. However, it ispreferred that there be no mutations, if any, little in the vicinity ofthe 3'-terminals of the primers which seem to greatly affect efficaciesof the elongation reaction of the primers. It is more preferred thatthere be mutations in the vicinity of the 5'-terminals of the primers.

In order to detect the four species of plasmodia using the nucleotidefragments according to the present invention as primers or probes, it ispreferred that these nucleotide fragments be used in the form of labeledfragments or solid carrier binding site-introduced fragments.

The labeled products of the nucleotide fragments according to thepresent invention include those obtained by binding a detectable labelto the above-described nucleotide fragments. In the present invention,either non-radioactive substances or radioactive substances may beavailable as the label, with the non-radioactive substances beingpreferred. Examples of the non-radioactive substances includefluorescent substances for example, fluorescein and its derivatives(fluorescein isothiocyanate and the like), rhodamine and its derivatives(tetramethylrhodamine isothiocyanate, texasred and the like)!,chemiluminescent substances (for example, acridine and the like),delayed fluorescence-emitting substances (DTTA: product of PharmaciaCo.), which can be directly detected.

When substances specifically binding to the above-mentiabove-mentionedlabels are utilized for detecting these labels, the labels can beindirectly detected. Biotin or some haptens are mentioned as labels insuch a case. In the case where biotin is used, avidin or streptavidinwhich specifically couples to biotin can be used. In the case where thehaptens are used, antibodies which specifically couple to the haptenscan be used. Compounds having a 2,4-dinitrophenyl group and digoxigenin,and besides biotin and fluorescent substances can be used as haptens.These labels may be introduced into the primers either singly or, ifnecessary, in various combinations with one another in accordance withthe conventionally-known methods (see Japanese Patent ApplicationLaid-Open Nos. 93098/1984 and 93099/1984).

The solid carrier binding site-introduced derivatives of the nucleotidefragments according to the present invention include those obtained byintroducing sites capable of coupling to a solid carrier into thenucleotide fragments according to the present invention. For example,the above-mentioned non-radioactive labels may also be used as the sitescapable of coupling to the solid carrier (solid carrier binding sites).Preferable examples thereof include biotin, fluorescent substances suchas fluorescein, and haptens such as compounds having a 2,4-dinitrophenylgroup and digoxigenin. These sites may be introduced into the nucleotidefragments according to the present invention in advance either singlyor, if necessary, in various combination with one another. In order toselectively bind the site to the solid carrier, the substance for thesite may preferably be different from that for the label. The solidcarrier in the present invention should be inactive against solvents andall reagents to be used in reactions, can be separated from solutions ofthe reagents by any known method and is able to selectively couple tothe above-described sites. Examples of such a solid carrier includesolid materials such as microtiter plates, polystyrene balls, agarosebeads and polyacryl beads, in which streptavidin, an antibody or thelike capable of trapping the above sites has been introduced. Themicrotiter plates are preferred because they are excellent in operatingcharacteristics, mechanization and the like.

For example, a carrier in which streptavidin has been introduced can beused in order to trap a product formed by a gene-amplifying reactionmaking use of a primer in which biotin has been introduced. Besides, inorder to trap a product formed by a gene-amplifying reaction making useof a primer in which a fluorescein residue or a 2,4-dinitrophenyl grouphas been introduced, a carrier in which its corresponding antibody hasbeen introduced can be used.

In order to detect the plasmodia with these probes, a dot blothybridization technique, a Southern hybridization technique or the likemay be used Hames, B. D., Nucleic Acid Hybridization, IRL Press (1985)!.As a target for the detection, not only DNA but also RNA may be used. Inthe present invention, 18S ribosome RNA, which is large in copy numberper cell, can be used as a target to enhance sensitivity. When thedetection must be conducted with still higher sensitivity, variousgene-amplifying reactions can be used. When products formed by thesereactions are used as targets, the sensitivity can be enhanced by leapsand bounds. For example, there may be conducted dot blot hybridizationor reverse dot hybridization in which PCR is adopted as thegene-amplifying reaction, and a product formed by the PCR is used as atarget Innis, M. A., PCR. Protocols Academic Press (1990)!. Further,when high-sensitive and easy detection is required, a method making useof a microtiter plate as described in Japanese Patent ApplicationLaid-Open No. 182035/1991 is more practical.

The detection of the four species of plasmodia by the gene-amplifyingreactions making use of the nucleotide fragments according to thepresent invention is basically performed in the following manner.

In the case where the four species of plasmodia may not be distinguishedfrom one another, that is, one wants to determine whether he is infectedwith a plasmodium or not, it is only necessary to conduct agene-amplifying reaction with a primer selected from the sequences (SEQID NOS:7) or a primer selected from the base sequence represented by(SEQ ID NO:2) and then to determine the presence of a product of theintended size formed by the gene-amplifying reaction by agarose gelelectrophoresis or the like. More simply, as described in JapanesePatent Application Laid-Open No. 252300/1989, one primer in which alabel has been introduced, and the other primer in which a solid carrierbinding site has been introduced may be used to conduct agene-amplifying reaction, and the resulting amplified product may bedetected by a reaction like ELISA (enzyme linked immunosorbent assay).

On the other hand, in the case where the four species of plasmodia mustbe distinguished from one another, it is possible to make a good use ofthe fact that the chain lengths of products formed by gene-amplifyingreactions with primers of (SEQ ID NOS:1, and 3-6) or primers selectedfrom the base sequence represented by (SEQ ID NOS:1, and 3-6) delicatelyvary according to the species of malaria, or that when a product formedby a gene-amplifying reaction is subjected to a treatment with arestricted enzyme, a fragment inherent in each of the malaria species isformed. A simpler method is a method in which gene-amplifying reactionsare conducted with primers represented by (SEQ ID NO:7-10) or primersselected from the base sequence represented by (SEQ ID NO:2), and theresulting amplified products are used as targets to conduct detectionwith probes represented by (SEQ ID NOS:1, and 3-6) or probes selectedfrom the base sequence represented by (SEQ ID NO:2). For example, thereis a method in which a gene-amplifying reaction is conducted with aprimer selected from (SEQ ID NOS:6-10) or a primer selected from thebase sequence represented by the sequence (SEQ ID NO:6-1), in which alabel has been introduced, and a product formed by this gene-amplifyingreaction is hybridized with each of probes of (SEQ ID NO:1, and 3-6),which have been immobilized to wells of microtiter plates, therebymaking detection.

The operation of the detection method according to the present inventionwill hereinafter be described in detail.

(1) Pretreatment of specimen

There are first provided specimens to be detected for the presence ofthe target plasmodium. Examples of the specimens principally includeblood obtained from infected persons or patients. The hemocytes of theblood collected can be lysed with a surfactant or the like for example,saponin, Biochem. Biophys. Res. Commun., 175, 179-184 (1991)! after itsserum component is removed or as it is, thereby removing the hemocytesby centrifugation. With respect to the plasmodium from which thehemocytes have been removed, its cells can be lysed by a treatment witha proteinase like the ordinary cells.

For the sake of simplified operation, it is necessary to omit thecentrifugation. In this case, care must be taken because a DNApolymerase reaction tends to be extremely inhibited by the bloodcomponents. For example, the whole blood may be treated with theabove-mentioned saponin and then treated with a proteinase or the likeas it is, thereby providing a sample for gene amplification. In thiscase, however, it is preferred that the pH of the blood after thecell-lysing reaction be measured in advance to add buffer as describedabove upon the cell-lysing treatment or gene-amplifying reaction so asto give a pH most suitable for the DNA polymerase reaction. When thewhole blood is subjected to the lysing treatment, its pH is lowered.Therefore, it is preferred that the pH and concentration of the bufferto be used be kept higher than those generally used in cell lysis andgene-amplifying reaction in order to optimize the pH in the DNApolymerase reaction.

(2) Gene-amplifying reaction

If the target plasmodium is present in the specimen, a gene-amplifyingreaction according to the elongation reaction of primers may be carriedout by adding the primers according to the present invention to thespecimen.

The elongation reaction of a primer can be carried out by introducingfour kinds of nucleoside triphosphates deoxyadenosine triphosphate,deoxyguanosine triphosphate, deoxycytidine triphosphate and thymidinetriphosphate (a mixture thereof may be referred to as dNTP)! assubstrates into the primer.

In the elongation reaction, E. coli DNA polymerase I, Klenow fragmentfrom E. coli DNA polymerase I, T4 DNA polymerase and the like are used.In particular, when a heat-resistant enzyme such as a Tag polymerase,which causes the elongation reaction at a high temperature, is used,specificity of primer's recognition of a target sequence can be enhanced(refer to Japanese Patent Application Laid-Open Nos. 314965/1989 and252300/1989 for further particulars).

The 18S ribosome RNA gene of the target plasmodium can be effectivelyamplified by repeating the elongation reaction using a combination forexample, a combination of the primer (SEQ ID NO:7) with the primer (SEQID NO:8), a combination of the primer (SEQ ID NO:9) with the primer (SEQID NO:10), a combination of the primer (SEQ ID NO:7) with the primer(SEQ ID NO:10) or a combination of the primer (SEQ NO:9) with the primer(SEQ ID NO:8)! of two kinds of the primers according to the presentinvention.

Further detailed procedures of the gene-amplifying reaction should bereferred to reference books "Jikken Igaku (Experimental Medicine), 8,No. 9 (1990), Yodosha, and "PCR Technology", Stockton Press (1989)!.

(3) Detection

Detection of a conjugate of the target gene with a detection primerproduced by a primer elongation reaction leads to detection of theplasmodium in the specimen. A preferred detection method variesaccording to the kind or form of the above-mentioned primer elongationproduct, i.e., a synthesized nucleic acid.

In general, the product formed by the gene-amplifying reaction forms adouble stranded DNA. Therefore, this synthesized nucleic acid can bedetected by electrophoresis Saiki, R. K., Science, 230, 1350-1354(1985)!, dot hybridization making use of labeled probes Saiki, R. K.,Nature, 324, 163-166 (1986)! or the like.

When a label has been introduced into a synthesized nucleotide chain,the nucleic acid can also be detected by reverse dot hybridizationSaiki, R. K., Proc. Natl. Acad. Sci. U.S.A., 86, 6230-6234 (1989)!, inwhich the labeled and synthesized nucleotide chain is hybridized with aprobe immobilized on a solid carrier, a column method making use of asolid adsorbent (see Japanese Patent Application Laid-Open No.314965/1989) or the like.

The nucleic acid can be detected with greater ease according to themethod described in Japanese Patent Application Laid-Open No.252300/1989. That is, there is a method in which a combination of thesolid carrier binding site-introduced derivative of the nucleotidefragment with the labeled fragment is used as a primer, a product formedby the gene-amplifying reaction is brought into contact with a solidcarrier, and impurities are then removed by washing with an appropriatesolvent. Even in this method, the chain of the synthesized nucleic acidelongated by the primer having the solid carrier binding site forms adouble stranded DNA with another synthesized nucleic acid elongated bythe labeled primer. Therefore, the target product formed by thegene-amplifying reaction is immobilized on the solid carrier in the formbearing the label thereon, resulting in specific fixation.

When a plasmodium is detected in distinction from other plasmodia, it isvery simple and practical to use the method described in Japanese PatentApplication Laid-Open No. 227998/1993. That is, there is a method inwhich the non-radioactive label-introduced fragment is used as a primerto conduct a gene-amplifying reaction, and the resulting reactionproduct is hybridized with the nucleotide fragments of (SEQ ID NO:6-10)immobilized to wells of microtiter plates. Only when a sequencecomplementary to a nucleotide fragment immobilized to the wells of amicrotiter plate exists, the product formed by the gene-amplifyingreaction is bound to the microtiter plate. In this case, it is onlynecessary to detect the label bound to the primer.

The detection of the label may be performed by any conventional methodaccording to the label used. For example, in the case where the label isa radioisotope, it is only necessary to measure the radioactivitythereof as it is. In the case where the label is biotin or a hapten, thesubstrate may be reacted with an avidin (or streptavidin)-enzymeconjugate or an antibody-enzyme conjugate, respectively, therebyobtaining a component detectable by a staining or fluorescent means.Besides, for example, in the case where the label is a fluorescentsubstance, it is only necessary to measure the fluorescent intensity bymeans of a fluorometer as it is.

EXAMPLES

The present invention will hereinafter be described in detail withreference to the following examples. However, this invention is notintended to be limited to these examples only.

Example 1

Synthesis of deoxyoligonucleotide and primer:

First of all, an oligonucleotide was synthesized on the basis of thephosphoamidite method of Caruthers et al. Tetrahedron Lett., 22, 1859(1981)! by means of a DNA synthesizer Model 318A manufactured by AppliedBiosystems Co. on the scale of 0.2 micromole.

On the other hand, an oligonucleotide in which a label or a solidcarrier binding site was introduced was obtained by synthesizing anoligonucleotide in the 5'-terminal of which an aminoalkyl group wasintroduced and then introducing the label or the solid carrier bindingsite in the oligonucleotide with an appropriate reagent. Examples ofthese syntheses are described below.

First, an oligonucleotide (5'GAACGAAAGTTAAGGGAGT; SEQ ID NO: 7) in the5'-terminal of which an aminoalkyl group was introduced was synthesizedby means of the DNA synthesizer Model 318A manufactured by AppliedBiosystems Co. A protective mononucleotide phosphoamidite wassuccessively added to 0.2 micromole of a support to add a base to the5'-terminal, and Aminolink II (trade name, Applied Biosystems Co.) wasthen added further. The addition product thus obtained was thenseparated from the support by a treatment with concentrated aqueousammonia, and the protecting group was removed.

After the deblocking, the thus-obtained product was subjected to gelfiltration through Sephadex G-50. Fractions of the peak eluted firstwere collected and concentrated. A 1M aqueous solution of NaHCO₃ (10μl), H₂ O (30 μl) and a solution of biotin N-hydroxysuccinimide ester(BRL Co., Ltd) in DMF (20 μg/μl, 50 μl) were then added to thethus-obtained aqueous solution of the aminoalkylated oligonucleotide (1O.D.: 10 μl). After mixing them, the mixture was allowed to stand atroom temperature. After 4 hours, the mixture was passed through a gelfiltration column (Sephadex G-50), and elution was effected with 50 mMTEAB buffer (a buffer solution of triethylammonium bicarbonate, pH 7.5).Fractions of the peak eluted first were collected to dry into solid.This sample was then purified by reverse-phase HPLC (column: μ-BondapakC18, eluent: 5-20% acetonitrile-50 mM triethylammonium acetate, pH 7.0,yield: 0.35 O.D.).

An oligonucleotide (5'ACTGAAGGAAGCAATCTAA; SEQ ID NO:8) in the5'-terminal of which a dinitrophenyl group (DNP) was introduced wasobtained by first synthesizing an oligonucleotide in which an aminoalkylgroup was introduced as in the case of the biotinylated oligonucleotideand using this as a starting material. To a solution of theaminoalkylated oligonucleotide (1.0 O.D., 180 μl) roughly purified bygel filtration, was added 1M NaHCO₃ (20 μl), to which a 5% (v/v) ethanolsolution of dinitrofluorobenzene (100 μl) was then added. The resultantmixture was heated at 37° C. for 2 hours. After completion of thereaction, the reaction mixture was subjected to gel filtration in thesame manner as in the biotinylated oligonucleotide to remove reagents,and purified by reverse-phase HPLC (yield: 0.38 O.D.).

Example 2

Determination of base sequence of 18S ribosome RNA gene of Plasmodiumovale (ovale malaria parasite):

Genes of an ovale malaria parasite were extracted from the blood of apatient who had been judged to be infected with the ovale malariaparasite from the observation of a blood smear. The blood (5 ml)containing the ovale malaria parasite was first suspended in PBS, andthe suspension was centrifuged for 10 minutes at 3,000 rpm. Thesupernatant was removed, and the resultant hemocytes were suspended in a0.15% (w/v) PBS solution of saponin (7.5 ml), and the suspension wasleft at rest for 20 minutes at 37° C. PBS was then added to 45 ml, andthe dilute suspension was centrifuged for 15 minutes at 3,000 rpm. Thesediment was suspended in PBS (45 ml), and the suspension wascentrifuged similarly. This process was further repeated twice. Thethus-obtained sediment was suspended in PBS (50 μl) and transferred to a2-ml centrifuge tube. This suspension was centrifuged for 5 minutes at10,000 rpm. The supernatant was removed, and the sediment was suspendedin a mixture (1.5 ml) of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0) and0.1M NaCl. Further, 20% SDS (37.5 μl) and proteinase K (10 μg/μl, 30 μl)were added to the suspension, followed by end-over-end mixing at roomtemperature for 1 hour. The resulting mixture was left at stand for 16hours at 37° C. Saturated saline was added to this mixture in an amountthird of the volume of the mixture, followed by end-over-end mixing. Theresulting mixture was left at rest for 15 minutes at room temperatureand then centrifuged for 15 minutes at 15,000 rpm. Hereinafter, theresultant supernatant was treated in accordance with the conventionalprocedures (treatment with phenol, washing with chloroform,precipitation with ethanol) for DNA extraction. The DNA sediment finallyobtained was dried and dissolved in water (240 μl) to prepare a solutionof ovale malaria DNA.

A part of the 18S ribosome RNA gene of the ovale malaria parasite wassubjected to gene amplification. That is, 35 μl of a lysis solution 100mM Tris-HCl (pH 8.9), 1.5 mM MgCl₂, 80 mM KCl, 500 μg/ml of BSA, 0.1%(w/v) sodium cholate, 0.1% Triton X-100, 200 μg/ml of proteinase K,0.45% Tween 20 and 0.45% NP40!, and one drop of mineral oil were added,and the mixture was heated for 10 minutes at 95° C. To this mixture,were added 10 μl of a liquid PCR mixture biotin-labeled primer (SEQ IDNO:7) (Bio-5'GAACGAAAGTTAAGGGAGT, 5 μg/ml), primer (SEQ ID NO:8)(5'ACTGAAGGAAGCAATCTAA, 5 μg/ml), dATP, dGTP, dCTP and TTP (each 1 mM),50 mM Tris-HCl (pH 8.9), 80 mM KCl 1.5 mM MgCl₂, 0.1% Triton X-100, 0.1%(w/v) sodium cholate, 500 μg/ml of BSA, and 1 U Tth DNA polymerase! andthe above-prepared ovale malaria parasite DNA solution (5 μl) to performa gene-amplifying reaction by means of a thermal cycler (PJ2000,manufactured by Perkin Elmer Co.) under conditions of 94° C. for 30seconds, 50° C. for 60 seconds, and 72° C. for 60 seconds (15 cycles).The liquid reaction mixture (1 μl) was taken out and added to a lysissolution (39 μl) subjected to a heat treatment at 94° C. for 10 minutes,thereby performing a gene-amplifying reaction (25 cycles) under the sameconditions as described above. A part of the liquid reaction mixture wastaken out and determined by agarose gel electrophoresis. As a result, aproduct of about 140 bp was able to be identified.

Streptavidin-immobilizing beads (Dynabeads, M280 Streptavidin, productof Dynal Co., 20 μl) were then washed with a cleaning solution (10 mMTris-HCl, pH 7.5; 1 mM EDTA; 2M NaCl, 20 μl). These beads were suspendedin the cleaning solution (40 μl), and the above-prepared ovale malariaparasite DNA solution (5 μl) was added to the resultant suspension. Theresultant mixture was mixed and left at rest for 15 minutes at roomtemperature. The supernatant was removed, and the residue was washedwith the cleaning solution (40 μl). Then, 0.1M NaOH (10 μl) was added,and the resultant mixture was left at rest for 10 minutes at roomtemperature. The supernatant was taken out and neutralized with 0.33MHCl (3 μl) to provide Solution B. The residual beads were washed with0.1M NaOH (50 μl), the cleaning solution (40 μl), and 10 mM Tris-HCl (pH7.6) and 0.1 mM EDTA (pH 8.0, 50 μl), and suspended in water (8 μl) toprovide Suspension A.

The primer (SEQ ID NO:8) (5'4ACTGAAGGAAGCAATCTAA) and the primer (SEQ IDNO:7) (Bio-5'GAACGAAAGTTAAGGGAGT) were used for Solution A andSuspension B, respectively, to determine base sequences in accordancewith the dideoxy method.

Example 3

Preparation of probe immobilized to wells of microtiter plate:

Oligonucleotides represented by the following sequences (A) (ovalemalaria parasite), (B) (quartan malaria parasite) and (C) (common tofalciparum, tertian, quartan and ovale malaria parasites) weresynthesized, and their 5'-terminals were phosphorylated with a T4polynucleotide phosphorylase and ATP. Their corresponding twooligonucleotides were mixed to form double stranded fragments (SEQ IDNO:11-13). ##STR3##

These fragments were separately bound to each other with a T4 DNAligase, and the resultant conjugates were treated with a Klenow fragmentfrom E. coli DNA polymerase I and four kinds of deoxyribonucleosidetriphosphates to make them blunt ends. A plasmid, pUC-Sfix2 (JapanesePatent Application Laid-Open No. 190194/1990) was excised with arestriction enzyme, BamH I, and the resultant fragments were then madeblunt ends with the Klenow fragment. These DNA fragments were boundusing the T4 DNA ligase to transform E. coli DH5. The intended cloneswere selected by the restriction enzyme analysis. The thus-obtainedplasmid was excised with a restriction enzyme Sfi I, and fragmentscontaining repetition of each of the above sequences were purified andrecovered by using electrophoresis. A fragment excised with Sfi I andcontaining excessive repetition of the sequence were bound to a vectorwith the T4 DNA ligase. In this case, a fragment obtained by excisingpUC-Sfix2 with Sfi I was used as the vector. This conjugate was used totransform MV1184, and clones each containing about 60 units of (SEQ IDNOS:10-15) were separately selected by restriction enzyme analysis.

From these clones, single stranded DNAs were prepared with a helperphage M13KO7 in accordance with the conventional method Molecularcloning, 4.29-4.32, 2nd ed.!.

Each of the single stranded DNAs thus obtained was diluted to aconcentration of 1 μg/50 μl with a solution containing 10 mM Tris-HCl(pH 7.6) and 1 mM EDTA, and an equiamount of a buffer for immobilization1.5M NaCl, 0.3M Tris-HCl (pH 8.0), 0.3M MgCl₂ ! was added to the dilutesolution. The mixture was mixed and added to wells of a microtiter plate(Sepaplate 8F H type, Sumitomo Bakelite Co., Ltd.) in an amount of 100μl per well. The wells were covered and left at rest for 16 hours at 37°C. Thereafter, the solution was removed from the wells, and themicrotiter plate was dried at 37° C. for 30 minutes. UVStratalinker™2400 (product of Stratagene Co.) was then used to carry outirradiation of 500,000 μJ. After the irradiation, the microtiter platewas washed 3 times with a cleaning buffer 1M NaCl, 2 mM MgCl₂, 0.1MTris-HCl (pH 9.3), 0.1% Tween 20; 200 μl). The microtiter plate on whichthe single stranded DNA containing (SEQ ID NOS:10-11) was immobilized,the microtiter plate on which the single stranded DNA containing (SEQ IDNOS:12-13) was immobilized, and the microtiter plate on which the singlestranded DNA containing (SEQ ID NOS:14-15) was immobilized were providedas microtiter plates for detecting the ovale malaria parasite, thequartan malaria parasite and all of the four plasmodia, respectively.The microtiter plates were conserved at 4° C.

Example 4

Simultaneous detection of falciparum malaria parasite, tertian malariaparasite and ovale malaria parasite:

DNAs extracted from plasmodia were used as samples A: falciparum malariaparasite (0.22 ng/μl, 5 μl), B: tertian malaria parasite (DNAcorresponding to 10⁴ parasites isolated from a patient), C: ovalemalaria parasite (DNA solution obtained in Example 2, 5 μl), D: DNAobtained from leukocytes of the healthy human (100 ng/μl, 5 μl)!.Solutions of the DNAs were separately added to 40 82 l of a lysissolution 100 mM Tris-HCl (pH 8.9), 1.5 mM MgCl₂, 80 mM KCl, 500 μg/ml ofBSA, 0.1% (w/v) sodium cholate, 0.1% Triton X-100, 200 μg/ml ofproteinase K, 0.45% Tween 20 and 0.45% NP40!. After the mixtures weretreated at 60° C. for 20 minutes and at 94° C. for 10 minutes, 10 μl ofa liquid PCR mixture biotin-labeled primer (SEQ ID NO:7) and DNP-labeledprimer (SEQ ID NO:8) (each 5 μg/ml), dATP, dGTP, dCTP and TTP (each 1mM), 50 mM Tris-HCl (pH 8.9), 80 mM KCl, 1.5 mM MgCl₂, 0.1% TritonX-100, 0.1% (w/v) sodium cholate, 500 μg/ml of BSA, and 1 U Tth DNApolymerase! were added to the samples to carry out respectivegene-amplifying reactions (under conditions of 94° C. for 30 seconds,50° C. for 60 seconds and 72° C. for 60 seconds, and 30 cycles).

After completion of the reactions, each 10 μl of the liquid reactionmixtures were added to streptavidin-immobilized wells of a microtiterplate, to which an alkaline phosphatase-labeled anti-DNP antibodydiluted with 100 μl of a buffer 50 mM Tris-HCl (pH 7.5), 0.15 mM MgCl₂,0.05% Tween 20! had been added in advance. After left over for 30minutes at room temperature, the wells were washed with about 300 μl ofthe above-described buffer. This washing process was repeated furthertwice, and 100 μl of a solution of p-nitrophenylphosphate (dissolved in4 mg/ml of mg/ml of a diethanolamine buffer) was added to the wells.After left over for 30 minutes at 25° C., the absorbance at 405 nm as tothe respective wells was measured by a microplate reader. As a result,all of the plasmodia were able to be detected by using the primersaccording to the present invention as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Simultaneous detection of falciparum,                                         tertian and ovale malaria parasites                                           Falciparum   Tertian     Ovale     Healthy                                    malaria      malaria     malaria   human                                      parasite (A) parasite (B)                                                                              parasite (C)                                                                            (D)                                        ______________________________________                                        Found  0.67      0.97        0.44    0.08                                     ______________________________________                                    

Example 5

Simultaneous and distinctive detection of falciparum malaria parasite,tertian malaria parasite and ovale malaria parasite:

After the same samples A, B, C and D as those used in Example 4 weretreated with the lysis solution in the same manner as in Example 4, 10μl of a liquid PCR mixture biotin-labeled primer (SEQ ID NO:9)(Bio-5'TCAGATACCGTCGTAATCTT) and biotin-labeled primer (SEQ ID NO:10)(Bio-5'CCAAACACTTTGATTTCTCAT) (each 5 μg/ml), dATP, dGTP, dCTP and TTP(each 1 mM), 50 mM Tris-HCl (pH 8.9), 80 mM KCl, 1.5 mM MgCl₂, 0.1%Triton X-100, 0.1% (w/v) sodium cholate, 500 μg/ml of BSA, and 1 U Tthpolymerase! were added to the samples to carry out respectivegene-amplifying reactions (under conditions of 94° C. for 30 seconds,50° C. for 60 seconds and 72° C. for 60 seconds, and 30 cycles). Aftercompletion of the reactions, each of the liquid reaction mixtures wassubjected to thermal denaturation for 5 minutes on a boiling water bath,and then quenched on an ice bath. Each 10 μl of the liquid mixture wereadded to wells of the microtiter plates for detecting the falciparummalaria parasite, the tertian malaria parasite, the ovale malariaparasite, the quartan malaria parasite and all of the four plasmodia,respectively, to which a solution for hybridization (5×SSC: 10 μl/well)had been added in advance, thereby performing hybridization at 60° C.for 1 hour. The hybridization solution was removed from the wells, andthe wells were washed 3 times with 2×SSC (200 μl/well). To the wellsthus washed, was added a streptavidin-alkaline phosphatase solutiondiluting streptavidin-alkaline phosphatase produced by BRL Co. to 1/2000with 0.1M Tris-HCl (pH 7.5), 0.3M NaCl and 2 mM MgCl₂, 0.05% (v/v)Triton X-100; 100 μl/well!. The thus-treated microtiter plates were leftover for 15 minutes at room temperature. The liquid reaction mixtureswere removed from the respective wells, and the wells were washed 3times with a cleaning solution 0.1M Tris-HCl (pH 7.5), 0.3M NaCl, 2 mMMgCl₂, 0.05% (v/v) Triton X-100; 200 μl/well). After the washing, asolution of p-nitrophenylphosphate (in 1M diethanolamine (pH 9.8) and0.5 mM MgCl₂ ; 4 mg/ml; 100 μl/well) was added to the wells to conductreaction at 25° C. for 30 minutes, and the absorbance at 405 nm as tothe respective wells was measured. As a result, all of the fourplasmodia were able to be simultaneously detected in distinction fromone another by using the primers according to the present invention asshown in Table 2.

                                      TABLE 2                                     __________________________________________________________________________    Simultaneous and distinctive detection of falciparum, tertian and ovale       malaria parasites                                                                             Sample                                                                        Falciparum                                                                          Tertian                                                                             Ovale Healthy                                                     malaria                                                                             malaria                                                                             malaria                                                                             human                                       Well            parasite (A)                                                                        parasite (B)                                                                        parasite (C)                                                                        (D)                                         __________________________________________________________________________    Found                                                                             Well for detection of                                                                     1.43  0.00  0.00  0.03                                            falciparum malaria parasite                                                   Well for detection of                                                                     0.01  1.82  0.00  0.02                                            tertian malaria parasite                                                      Well for detection of                                                                     0.00  0.00  0.66  0.00                                            ovale malaria parasite                                                        Well for detection of                                                                     0.00  0.00  0.00  0.00                                            quartan malaria parasite                                                  __________________________________________________________________________

According to the present invention, all of the plasmodia (falciparum,tertian, quartan and ovale malaria parasites), which infect the human,can be detected easily, rapidly and with a high sensitivity ordistinguished accurately from one another. Therefore, the invention isuseful in treatment for malaria and large-scale mass examination in thearea where malaria is prevalent.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 18                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: P. ovale                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GAAAATTCCTTTCGGGGA18                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: P.ovale                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGTTTTGGATGAAACATTTTT60                AAATAAGAAAATTCCTTTCGGGGAAATTTC90                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: P. malariae                                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAGACATTCTTATATATG18                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: P. falciparum                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AAGTCATCTTTCGAGGTGAC20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: P. vivax                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAATTTTCTCTTCGGAGTTTA21                                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CGACTAGGTGTTGGATGA18                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GAACGAAAGTTAAGGGAGT19                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ACTGAAGGAAGCAATCTAA19                                                         (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCAGATACCGTCGTAATCTT20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: YES                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: P. falciparum                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CCAAAGACTTTGATTTCTCAT21                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TATTGAAAATTCCTTTCGGGGA22                                                      (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TATTGAGACATTCTTATATATG22                                                      (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "Synthetic DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TTATCGACTAGGTGTTGGATGA22                                                      (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AATCAAGAACGAAAGTCGGAGGTTCGAAGACGATCAGATACCGTCGTAGTTCCGACCATA60                AACGATGCCGACCGGCGATGCGGCGGCGTTATTCCCATGACCCGCCGGGCAGCTTCCGGG120               AAACCAAAGTCTTTGGGTTCCGGGGGGAGT150                                             (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 182 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AATCAAGAACGAAAGTTAAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATA60                AACTATGCCGACTAGGTGTTGGATGAAAGTGTTAAAAATAAAAGTCATCTTTCGAGGTGA120               CTTTTAGATTGCTTCCTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGA180               GT182                                                                         (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 184 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      AATCAAGAACGAAAGTTAAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATA60                AACTATGCCGACTAGGCTTTGGATGAAAGATTTTAAAATAAGAATTTTCTCTTCGGAGTT120               TATTCTTAGATTGCTTCCTTCAGTGCCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGC180               GAGT184                                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 191 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AATCAAGAACGAAAGTTAAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATA60                AACTATGCCGACTAGGTTTTGGATGATAGTGTAAAAAATAAAAGAGACATTCTTATATAT120               GAGTGTTTCTTTTTAGATAGCTTCCTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTT180               CTGGGGCGAGT191                                                                (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      AGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGTTTTGGATGAAACATTTTT60                AAATAAGAAAATTCCTTTCGGGGAAATTTC90                                              __________________________________________________________________________

We claim:
 1. A nucleotide fragment consisting of a base sequencerepresented by the following sequence:

    5'GAAAATTCCTTTCGGGGA3'                                     (SEQ ID NO:1)

or the base sequence complementary thereto.
 2. A nucleotide fragmentconsisting of a base sequence represented by the following sequence:##STR4## or the base sequence complementary thereto.
 3. A nucleotidefragment consisting of a base sequence represented by the followingsequence:

    5'GAGACATTCTTATATATG3'                                     (SEQ ID NO:3)

or the base sequence complementary thereto.
 4. A method ofdistinguishing falciparum malaria, tertian malaria, quartan malaria andovale malaria from one another comprising hybridizing a nucleotidefragment consisting of a base sequence represented by one of thefollowing sequences:

    5'AAGTCATCTTTCGAGGTGAC3'                                   (SEQ ID NO:4)

    5'GAATTTTCTCTTCGGAGTTTA3'                                  (SEQ ID NO:5)

    5'GAGACATTCTTATATATG3'                                     (SEQ ID NO:3)

    5'GAAAATTCCTTTCGGGGA3'                                     (SEQ ID NO:1)

    5'CGACTAGGTGTTGGATGA3'                                     (SEQ ID NO:6)

or the base sequences complementary thereto to a sample DNA in whichfalciparum malaria, tertian malaria, quartan malaria or ovale malaria isto be detected, detecting binding of said nuclecide fragment to saidsample DNA, and correlating binding of said nucleotide fragment to saidsample DNA to the presence of one of falciparum malaria, tertianmalaria, quartan malaria or ovale malaria.
 5. Primers for detectingplasmodia each consisting of a nucleotide fragment consisting of the oneof the base sequences represented by the following sequences:

    5'GAACGAAAGTTAAGGGAGT3'                                    (SEQ ID NO:7)

    5'ACTGAAGGAAGCAATCTAA3'                                    (SEQ ID NO:8)

    5'TCAGTTACCGTCGTAATCTT3'                                   (SEQ ID NO:9)

and

    5'CCAAAGACTTTGATTTCTCAT                                    (SEQ ID NO:10).


6. A primer or probe for detecting an ovale malaria parasite consistingof a nucleotide fragment comprising a base sequence represented by thefollowing sequence:

    5'GAAAATTCCTTTCGGGGA3'                                     (SEQ ID NO:1),

the base sequence complementary thereto or a mutation sequence thereof,wherein said primer or probe specifically detects an ovale malariaparasite under the following conditions: (A) amplification in 100 mMTris-HCl (pH 8.9), 1.5 mM MgCl₂, 80 mM KCl, 500 μg/ml BSA, 0.1% (w/v)sodium cholate, 0.1% Triton X-100, 200 μg/ml of proteinase K, 0.45%Tween 20 and 0.45% NP40 under conditions of 94° C. for 30 seconds, 50°C. for 60 seconds, and 72° C. for 60 seconds; or (B) hybridization in5×SSC at 60° C. for 1 hour.
 7. A primer or probe for detecting an ovalemalaria parasite consisting of a nucleotide fragment comprising a basesequence represented by the following sequence: ##STR5## or the basesequence complementary thereto or a mutation sequence thereof, whereinsaid primer or probe specifically detects an ovale malaria parasiteunder the following conditions:(A) amplification in 100 mM Tris-HCl (pH8.9), 1.5 mM MgCl₂, 80 mM KCl, 500 μg/ml BSA, 0.1% (w/v) sodium cholate,0.1% Triton X-100, 200 μg/ml of proteinase K, 0.45% Tween 20 and 0.45%NP40 under conditions of 94° C. for 30 seconds, 50° C. for 60 seconds,and 72° C. for 60 seconds; or (B) hybridization in 5×SSC at 60° C. for 1hour.